Computational and in vitro analyses to identify the anticoagulant regions of Echicetin, a snake venom anticoagulant C-type lectin (snaclec): possibility to develop anticoagulant peptide therapeutics?

Abstract

Snake venom C-type lectins (Snaclecs) display anticoagulant and platelet-modulating activities; however, their interaction with the critical components of blood coagulation factors was unknown. Computational analysis revealed that Echicetin (Snaclec from Echis carinatus venom) interacted with heavy chain of thrombin, and heavy and light chains of factor Xa (FXa). Based on FXa and thrombin binding regions of Echicetin, the two synthetic peptides (1A and 1B) were designed. The in silico binding studies of the peptides with thrombin and FXa showed that peptide 1B interacted with both heavy and light chains of thrombin and, peptide 1A interacted with only heavy chain of thrombin. Similarly, peptide 1B interacted with both heavy and light chains of FXa; however, peptide 1A interacted only with heavy chain of FXa. Alanine screening predicted the hot-spots residues for peptide 1A (Aspartic acid6, Valine8, Valine9, and Tyrosine17 with FXa, and Isoleucine14, Lysine15 with thrombin) and peptide 1B (Valine16 with FXa). Spectrofluorometric interaction study showed a lower Kd value for peptide 1B binding with both FXa and thrombin than peptide 1A, indicating higher binding strength of the former peptide. The circular dichroism spectroscopy also established the interaction between thrombin and the custom peptides. The in vitro study demonstrated higher anticoagulant activity of peptide 1B than peptide 1A due to higher inhibition of thrombin and FXa. Inhibition of anticoagulant activity of the peptides by respective anti-peptide antibodies corroborates our hypothesis that peptides 1A and 1B represent the anticoagulant regions of Echicetin and may be developed as antithrombotic peptide drug prototypes.

Communicated by Ramaswamy H. Sarma

Keywords:

• Echicetin
• anticoagulant peptide
• Snaclec
• thrombin inhibitor
• FXa inhibitor
• antithrombotic drugs

Acknowledgments

K.R. and U.P. received DBT studentships and Ph.D. fellowships from Tezpur University, respectively. A.P. received a postdoctoral fellowship from IASST. A.K.M. thanks SAIC, IASST for the instrumental facility.The authors thank Dr. S. S. Ghosh and Ms. M. Saha, IIT, Guwahati for CD analysis. The authors thank Ms. S. Kalita, DST-Inspire JRF, Department of Chemistry, Tezpur University, for molecular dynamics simulation study.

Disclosure statement

The authors report there are no competing interests to declare.

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Funding

The author(s) reported there is no funding associated with the work featured in this article.